This presentation of my lecture, to Epidemiology students, briefs about different methods for differentiating or finding similarities among isolates of pathogens required establishing causal associations in epidemiological disease diagnosis.

1. Detection and Characterization of
Pathotypes, Serotypes, Biotypes,
Phenotypes, and genotypes
of Pathogens: An Integral Element in
Disease Investigation and Control
DOI: 10.13140/RG.2.2.25741.10727
Available at:
https://www.researchgate.net/publication/372787130_Detection_and_Characterization_of_Pathotypes_Serotypes_Bioty
pes_Phenotypes_and_genotypes_of_Pathogens_An_Integral_Element_in_Disease_Investigation_and_Control
Bhoj R Singh
Division of Epidemiology, ICAR-IVRI,
Izatnagar-243122, India
brs1762@gmail.com
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2. Definitions
• Pathotype: A population of a parasite/ pathogen species in which all individuals have a stated
pathosystem (host) character (pathogenicity or parasitic ability) in common. Though possible but rare to
have a single pathotype of pathogen species. The pathotypes are related to the pathogenicity potential of
a pathogen based on the presence of virulence factors.
• Virotype: A subset of individuals of a pathogen species sharing common virulence factors/ deterninats.
• Serotype/ Serovar: Group of individuals of a pecies having a distinct variation in antigenic/ immunogenic
epitopes within a species of bacteria or virus or of different individuals.
• Biotype/ Biovar: Biotypes is a populations within a species that differ in their ability to utilize a particular
trait as ability to use or produce specific biomolecule(s) in a particular genotype. Two biotypes may differ
jus through one codone change leading to observable change (phenotype) as one-codon change in
chloroplast GS, making it less sensitive to glufosinate.
• Toxinotype: A population of a species of pathogen producing specific type of toxin or a defined set of
toxins.
• Phenotype: Phenotype is the external or physical characteristics that appear in an organism. A
subpopulation in a species having a set of observable characteristics resulting from the interaction of
its genotype with the environment is defdined as a single phenotype.
• Genotype: A sub-population of a pathogen or a species having (a set of genes that it carries) common
DNA/ RNA markers represent a specific genotype. Some developed DNA marker methods are RFLP, RAPD,
AP-PCR, PFGE, AFLP and now WGS/ NGS. It is often called as Quasispecies theory or Darwinian Evolution
model, is primarily a theory of mutation-selection balance at extremely high mutation rates to separate it
from Classical population.
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3. Primary requirement for a pathogen to be
Characterization of Pathotypes, Serotypes,
Biotypes, Phenotypes, and genotypes
• Pure isolate/ culture
• Efficient system:
– Host for pathotyping, though now in-vitro methods are used but
they are just predict the pathotype not really determine the
pathotypes.
– Antibodies and antigenicity of the pathogen for serotyping. Now,
some genotypic methods are also in practice to predict serotype.
– Ability of the pathogen to grow in laboratory environment under
defined conditions for Biotyping.
– Integrity and Purity of the pathogen for Phenotyping
– Background knowledge of genotypes and suitable platform for
genotyping of an established/ known pathogen. For Novel
pathogens, a suitable genotyping platform as NGS.
• Expertise
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4. Pathotyping
Host: Experimental/ Model and real host/ Quasispecies models
Epidemiologically Hosts may be : Primary host, Secondary host (Intermediate), Paratenic host,
Accidental host, and Reservoir host etc.
• Primary host (a.k.a. definitive/final host): a host in which an organism partially/completely sexually
develops, and disease or effect of parasitism is apparent.
• Secondary host (intermediate host): a host in which an organism partially/completely asexually and may be
sexually developing without having a disease.
Both definitive and intermediate hosts are obligate to the life cycle of a parasite/ pathogen.
• Accidental host: a host that shelters an organism which does not usually parasitize that host
• Incidental host (a.k.a. dead-end host): a host that shelters an organism but is unable to transmit the
organism to a different host. They may permit development of a pathogen to some extent but not
permit development of its all statges.
• Partenic/ transport host: Paratenic hosts may span a wide range of fauna and are not needed in
the nematode's life cycle, but act as reservoirs in which different asexual/ larval stages of the
parasite/ Pathogen can persist but not develop further.
• Reservoir host: a host that shelters an organism without harm to itself. A pathogen hosts span a
wide range of fauna and are not needed in the pathogen's life cycle, but act as reservoirs in which
different stages of the pathogen can persist but not develop further.
• Mechanical host (fomites): No development/ growth of the pathogen but pathogen is just
mechanically carried from one host to another host like flies and insects transfer LSD.
• Symbionts: Members of relationship called symbiosis where both partners have benefits of living
together.
• Commensals: Commensalism is a type of symbiotic relationship where one partner
benefits whereas the second partner (the host) are neither helped nor harmed. The organism that
receives the refuge and nourishment is called the ‘commensal’. Most of the normal floras of the
human body can be considered as commensals.
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5. Pathotyping
• Experimental pathotyping: Understanding pathophysiology in Different types of
hosts and experimental model hosts. The Genetic make up of host and pathogen
boith decides the outcome and may not be true for the population in a defined
spatiotemporal situation.
• Observational studies : Case Controls/ Cohorts, always specific in spatotemorality.
Common pathovars of E. coli are Enterotoxigenic E. coli (ETEC),
Enterohaemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC),
Enteropathogenic E. coli (EPEC), Enteroadhesive E. coli (EAEC), and
Necrotoxigenic E. coli (NTEC).
• In Vitro studies: For determination of virulence (virotyping) and predicting
pathogenic potential. May be misleading and hypothetical because host-pathogen
interaction element is missing.
• Genotypic methods: detection of genetic markers of pathogenicity to predict a
pathotype. The egg-contaminating phenotype of Salmonella enterica serotype
Enteritidis was linked to single-nucleotide polymorphisms (SNPs) occurring in cyaA,
which encodes adenylate cyclase that produces cAMP and pyrophosphate from
ATP.
• Virotyping can be done using PCR methods, and also using in-vivo or in-vitro
determination of virulence factors of pathogens.
• HA/ Hly Typing: haemagglutination and Haemolysin typing requires detections of
haemagglutination and haemolysisis of different types of RBCs (from different host
species, tanned / fresh, in presence of Mannose etc.)
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6. Serotyping
Methods for determining serotype of a
pathogen Conventional and Genotypic
• Conventional: Using Polyclonal antibodies and
Using Monoclonal antibodies for detection of
specific ‘O’, ‘H’, ‘K’ and ‘F’ antigens.
• Serological Tests used: Agglutination/
Coagglutination/ Precipitation/ ELISA/ IHA/
CFA etc.
• Genotypic: Detection of genes through PCR
for specific ‘O’, ‘H’, ‘K’ and ‘F’ antigens.
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7. Biotyping
Biochemical and physiological methods are used for Biotyping of pathogens
There may be several biotypes within a serotype and vice-versa.
Can be done for culturable microbes.
• Common Biotyping methods:
– Production/ synthesis of specific biomoleculaes/ detectable (visually or with
aids) physiological traits (growth/ colour/ size)
– Utilization of specific biomolecules/ organic and inorganic chemicals.
– Degradation of specific biomolecules/ organic and inorganic chemicals.
– Resistance to different biomolecules/ organic and inorganic chemicals,
antibiograms are of common use
– Expression of specific detectable (visually or with aids) physiological traits
(growth/ colour/ size) in presence or absence of biomolecules/ organic and
inorganic chemicals.
– Requirement of specific biomolecules/ organic and inorganic chemicals for
physiological functions and/ or pathogenesis.
– Specific environmental conditions required for physiological functions and/ or
pathogenesis e.g. aerobic, microaerobic, anaerobic, capanophilic, acidophilic,
halophilic, photophilic, photophobic etc.
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8. Genotypig
• Can be done even for non-culturable microbes.
• The most fastest growing epidemiological tool
having the highest discretionary power,
consistency (repeatability), an vitro tool.
• Genotyping is becoming a potent in vitro tool for
biotyping, virotyping, serotyping and
pathotyping.
• Requires biotechnologically efficient laboratories
and expertise to use and understand the
outcome (results).
• Bioinformatics and sequencing (targeted or
whole genome) methods are the backbone of
genotyping.
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9. Methods in Genotyping
https://currentprotocols.onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.727
Partial genotyping methods (only a small fraction of an individual's genotype is determined)
• Restriction fragment length polymorphism (RFLP) of genomic DNA: IS6110-based restriction
fragment length polymorphism typing for MTB
• Random amplified polymorphism detection (RAPD)
• Amplicon polymorphism-PCR (AP-PCR)
• Pulse field gel electrophoresis (PFGE)
Targeted Genotyping
• Amplified fragment length polymorphism (AFLP), specific primers are used
• MLVA (Multiple‐Locus Variable number tandem repeat Analysis)
• ERIC PCR (Enterobacterial repetitive intergenic consensus (ERIC) sequences are 127-bp imperfect
palindromes that occur in multiple copies in the genomes of enteric bacteria and vibrios).
• Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeats
(MIRU-VNTR): polymerase chain reaction (PCR) amplification of a highly polymorphic direct repeat
locus in the M. tuberculosis genome.
• Allel specific oligonucleotide probe (ASO) hybridization
• Single nucleotide polymorphism (SNPs) using qPCR
• Copy Number Variation Analysis
• Pyrosequencing (allelic typing)
• (epi)GBS or RAD seq.
Complete Genotyping: Whole genome sequencing (WGS) possible through new generation sequencers
(NGS), Genome-Wide, Microarray of whole genome
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10. Other typing methods used for pathogens in
epidemiology
• Phage-typing
• Bacteriocin/ Colicin typing
• Antibiograms
• Fimbiae typing
• Morphotyping
• Multilevel Genome Typing
• Plasmid profiling and Genotyping of plasmids
• Whole Genome Multilocus Sequence Typing (wgMLST)
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11. Quiz
• Name biotypes of Salmonella and Gallibacterium having belonging to the same serotypes.
• Enlist the methods used in epidemiological genotyping of SARS CoV-2 and FMD viruses.
• Name the bacteria where toxinotyping is used for discerning epidemiologically different strains.
• Name the different pathotypes of
– Escherichia coli
– Clostridium perfingence
– Bacillus anthrecis
– Avian influenza virus
– SARS CoV-2
• Name the methods used for biotyping, and biotypes of
– Brucella melitensis and B. abortus
– Yersinia enterocolitica
– Salmonella Typhimurium and S. Gallinarum
– Bacillus cereus and B. subtilis
– Gallibacterium anatis
– Vibrio cholerae
– BVDV
• Name the methods of genotyping for
– Enterobacteriaceae members
– Vibrionaceae members
– Mycobacterium tuberculosis
– Brucella abortus
• Name the antigens and methods commonly used for serotyping of
– Pasteurella
– Salmonella
– Escherichia
– Klebsiella
– Streptococci
– Influenza viruses
– FMDV 11